7 replies to this topic

[pmaj]

Hello,

I was wondering if anyone has performed western blots for p53 and seen double bands for the protein. I get two bands very close to each other and I do not know what to make of it.

Pmaj

[bob1]

there are quite a few isoforms of p53, it could be that you are detecting one of these.  Which antibody are you using?  I find DO-1 the best.

[jiajia1987]

hey peeps,

I am sorry to add on to this. but I have been working mainly on evolving Mdm2. I will be working on p53 now as well.

Any good papers to recommend me to read?

And... my phd student was telling me that p53 binds non specifically to other random DNA as well other than their p53 response element, is this true?

[pmaj]

jiajia1987, on Sep 8 2009, 09:09 PM, said:

hey peeps,

I am sorry to add on to this. but I have been working mainly on evolving Mdm2. I will be working on p53 now as well.

Any good papers to recommend me to read?

And... my phd student was telling me that p53 binds non specifically to other random DNA as well other than their p53 response element, is this true?

There are tons of good papers to read when it comes to p53. As far as binding to random DNA, that is not really the case. p53 looks for a so called"consensus sequence" on its target and when it somewhat matches that sequence, it goes and executes the transcription of its target gene. Mdm2 is a special target that has a promoter within its first intron with 2 p53 recognition/response elements (REs) (aka the consensus sequence). The consensus sequence was first described by el-Deiry and c workers and it comprises of two half regions and each region consists of 10 nucleotide. This sequence is degenerate, which makes a lot of genes potential p53 targets.

With mdm2 the two p53 REs are slightly different from the consensus. this makes the binding of p53 a little loose. only when p53 is highly expressed, does it go and transcribe the mdm2 gene, so targets which match exactly with the sequence can be activated by basal levels of p53 protein, while the genes with some variation in the sequence need more p53 to get actively transcribed.


Following are the articles that might be of help to you:
1. A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome

2. Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity

3. The complexity of p53 modulation: emerging patterns from divergent signals

4. Regulating the p53 pathway: in vitro hypotheses, in vivo veritas

5. A functional p53 responsive intronic promoter is contained within the human mdm2 gene

6. Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential.

7. p53 binds to a constitutively nucleosome free region of the mdm2 gene

Hope this helps

Bests

Pmaj

[pmaj]

bob1, on Sep 8 2009, 08:23 PM, said:

there are quite a few isoforms of p53, it could be that you are detecting one of these.  Which antibody are you using?  I find DO-1 the best.

I am using  Cat # OP43L  Anti-p53 (Ab-6) (Pantropic) Mouse mAb (DO-1) from Calbiochem (EMD biosciences)

Pmaj

[s_laub]

I don't remember where I read this or whatever but I remember reading that getting two bands for p53 is something common?

[jiajia1987]

pmaj, on Sep 10 2009, 05:36 AM, said:

jiajia1987, on Sep 8 2009, 09:09 PM, said:

hey peeps,

I am sorry to add on to this. but I have been working mainly on evolving Mdm2. I will be working on p53 now as well.

Any good papers to recommend me to read?

And... my phd student was telling me that p53 binds non specifically to other random DNA as well other than their p53 response element, is this true?

There are tons of good papers to read when it comes to p53. As far as binding to random DNA, that is not really the case. p53 looks for a so called"consensus sequence" on its target and when it somewhat matches that sequence, it goes and executes the transcription of its target gene. Mdm2 is a special target that has a promoter within its first intron with 2 p53 recognition/response elements (REs) (aka the consensus sequence). The consensus sequence was first described by el-Deiry and c workers and it comprises of two half regions and each region consists of 10 nucleotide. This sequence is degenerate, which makes a lot of genes potential p53 targets.

With mdm2 the two p53 REs are slightly different from the consensus. this makes the binding of p53 a little loose. only when p53 is highly expressed, does it go and transcribe the mdm2 gene, so targets which match exactly with the sequence can be activated by basal levels of p53 protein, while the genes with some variation in the sequence need more p53 to get actively transcribed.


Following are the articles that might be of help to you:
1. A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome

2. Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity

3. The complexity of p53 modulation: emerging patterns from divergent signals

4. Regulating the p53 pathway: in vitro hypotheses, in vivo veritas

5. A functional p53 responsive intronic promoter is contained within the human mdm2 gene

6. Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential.

7. p53 binds to a constitutively nucleosome free region of the mdm2 gene

Hope this helps

Bests

Pmaj

wow.. this is GREAT information! thanks so much! I always have difficulty trying to find good papers online. I am usually not very sure of how I should go about finding the papers!

[pmaj]

bob1, on Sep 8 2009, 08:23 PM, said:

there are quite a few isoforms of p53, it could be that you are detecting one of these.  Which antibody are you using?  I find DO-1 the best.

Also the DO-1 antibody is pantropic, meaning that it binds indiscriminately. since p53 shares some homology between its isoforms, do you think I might be seeing either p63 or 73?